In previous experiments, we have demonstrated that the SV40 late promoter could be trans-activated by simian virus 40 (SV40) T-antigen in the absence of DNA replication. Activation could be achieved by either cotransfection of the late promoter with a plasmid coding for T-antigen or by transfection of the late promoter into COS-1 cells which constitutively express T-antigen. In the latter case, it was not clear whether expression of the endogenous T-antigen was continuously required or whether a set of cellular transcription factors alone led to the activation of the late promoter. To test these alternatives, we have performed transfection experiments in ts2 COS cells, which express the ts 1609 SV40 T-antigen. Transfection at the non-permissive temperature (40 degrees C) resulted in 5- to 10-fold reduction in SV40 late promoter activity compared to the permissive temperature (32 degrees C). An in vitro transcription system has been developed in order to study the mechanism of late promoter activation. Manley whole cell extracts were prepared from the trans-activation-positive COS-1 cell line. Preincubation of an SV40 DNA template with the COS-1 extract preferentially increases transcription from the major late start site at m.p. 325. Activation of the late promoter required optimization of the DNA template concentration ratio of COS-1 and HeLa extracts, and the length and temperature of preincubation. Under these conditions, increased transcription from the other SV40 late initiation sites or from the adeno major late promoter was not observed. The promoter sequence requirements for activation of the SV40 late promoter and the requirement for SV40 T-antigen in the preincubation COS-1 extract are presently being analyzed.